Cow mil (CM) is one of the major causes of food hypersensitivity in children. Cow milk allergy (CMA) is associated with a broad spectrum of IgE-mediated and non-IgE-mediated hypersensitivity disorders. While the clinical features of IgE-mediated CMA reactions are generally expressed as classic "immediate" reactions, the basic immunopathogenic mechanisms involved in IgE and cell-mediated systemic and gastrointestinal reactions are poorly understood. One of the major difficulties in studying CMA and other food allergies, is the lack of suitable animal models. A major barrier to the generation of food allergic mouse models is the early development of oral tolerance. We have recently developed a mouse model of IgE-mediated CMA utilizing intragastric (ig) sensitization and challenge of 3-week-old C3H/HeJ mice with whole CM by using the adjuvant cholera toxin (CT) to overcome oral tolerance. We will further characterize and validate this model of immediate hypersensitivity to CM. Because previous studies, including our own, suggest that age at first feeding may be a determining factor in sensitization to dietary proteins in mice as well as in humans, we will attempt to generate a "natural" non-adjuvant dependent mouse model of CMA by using neonatal mice sensitized with CM in the absence of CT. The use of our current model together with this "natural" model of CMH will allow us to accomplish the Specific Aims of this proposal. In parallel with Project #1, we will determine the IgE- and IgG2-binding B-cell epitopes and T-cell epitopes of casein. We will use our models to investigate the mechanisms involved in the regulation and oral tolerance to both milk and a control soluble protein. Cytokine (Th1/Th2) expression and T cell phenotype (CD4+/CD8+) will be determined. We will also determine whether a lack of oral tolerance to CM is associated with altered intestinal permeability. This aim will be tightly linked to studies in Project #3. Moreover, in coordination with Project #2, we will determine the site of antigen entry in both cow milk allergic mice and their normal control littermates. The intracellular trafficking pattern as well as the kinetics of uptake will be assessed using electron and confocal microscopy. Accomplishing these goals will provide a basis for understanding basic mechanisms involved in CMA and for exploring novel therapeutic approaches to control CMA.